Microfluidic testing system with cell capture/analysis regions for processing in a parallel and serial manner

ABSTRACT

A microfluidic chip system includes an input for receiving the biologic sample, and a first reading window for enabling a detection of the biologic material within the biologic sample. A first plurality of pathways is provided each for determining a treatment agent providing a best treatment efficacy for the predetermined biologic material. A first micro-pump is provided for pumping a portion of the biologic sample into each of the first plurality of pathways. A second plurality of pathways is provided, each for determining a dosage level of a particular one of the plurality of treatment agents with respect to the predetermined biologic material. A plurality of second micro-pumps are provided for pumping a second portion of the biologic sample into a selected one of the second plurality of pathways responsive to the determination of treatment efficacy of the treatment agent providing a best treatment of the predetermined biologic material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/186,505, filed Nov. 10, 2018, and entitled MICROFLUIDIC TESTINGSYSTEM WITH CELL CAPTURE/ANALYSIS REGIONS FOR PROCESSING IN A PARALLELAND SERIAL MANNER, which claims priority to and the benefit of U.S.Provisional Application No. 62/584,651, filed Nov. 10, 2017, andentitled MICROFLUIDIC TESTING SYSTEM WITH CELL CAPTURE/ANALYSIS REGIONSFOR PROCESSING A PARALLEL AND SERIAL MANNER, the contents of which areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention pertains in general to a microfluidics lab-on-chipsystem and, more particularly, to the use of a microfluidics chip andtesting at the point of care.

BACKGROUND

The emergence and spread of antibiotic-resistant bacteria are aggravatedby incorrect prescription and use of antibiotics. Courts have thisproblem is the fact that there is no sufficiently fast diagnostic testto guide correct antibiotic prescription at the point of care.Currently, some fluid sample is retrieved from a patient and forwardedto a lab for testing to determine a specific treatment regimen. As asafeguard, the patient is sometimes initially given large doses of ageneral antibiotic until a more specific antibiotic can be determined totarget the specific bacteria. This can take upwards of two or threedays, as the process requires growing the bacteria in some culturemedium and observing its response to various antibiotics.

SUMMARY

The present invention disclosed and claimed herein, in one aspect,comprises a microfluidic chip system for testing a treatment agent for apredetermined biologic material. The system includes an input forreceiving the biologic sample, the biologic sample containing thepredetermined biologic material that must be treated via one of aplurality of treatment agents. A first reading window this provided forenabling a detection of the predetermined biologic material within thebiologic sample. A is cell counter associated with the reading windowfor applying a tagging agent to cells of the detected biologic materialwithin the biologic sample. A first reservoir is provided for holdingthe biologic sample containing the predetermined biologic materialhaving the tagging agent applied thereto. A first plurality of pathwaysis provided each for determining a treatment agent of the plurality oftreatment agents providing a best treatment efficacy for thepredetermined biologic material within the biologic sample. A firstmicro-pump this provided for pumping a portion of the biologic sampleinto each of the first plurality of pathways. A second plurality ofpathways is provided, each for determining a dosage level of aparticular one of the plurality of treatment agents with respect to thepredetermined biologic material. A plurality of second micro-pumps areprovided for pumping a second portion of the biologic sample into aselected one of the second plurality of pathways responsive to thedetermination of treatment efficacy of the treatment agent providing abest treatment of the predetermined biologic material.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding, reference is now made to thefollowing description taken in conjunction with the accompanyingDrawings in which:

FIG. 1 illustrates a high-level view of a microfluidics chip of thepresent disclosure;

FIGS. 2A-2C illustrate detailed views of the multiple stages of analysisprovided by the microfluidics chip of FIG. 1;

FIGS. 3A-3D illustrate diagrammatic views of the various cell captureregions and the interspersed pumps for the microfluidics chip of FIG. 1;

FIGS. 4A-4G illustrate detailed views of the first viewing stage;

FIG. 5 illustrates a detailed view of the first parallel driving stage;

FIGS. 5A-5B illustrate details of the coating applied to the microchannels in the first driving stage;

FIG. 6 illustrates a detail of the serial driving stage;

FIGS. 7A-7D illustrate detailed views of a valveless nozzle/diffusermicropump;

FIG. 8 illustrates a detailed view of a piezoelectric micropump;

FIG. 9 illustrates a detailed view of a multi-chamber micropump withcheck valves;

FIG. 10 illustrates a flowchart for the high-level operation of themicrofluidics chip;

FIG. 11 illustrates a flowchart for the initial loading operation of thefluid sample;

FIG. 12 illustrates a flowchart for the viewing or cell counter stage ofanalysis;

FIGS. 13A-13C illustrate diagrammatic use for the cell counter;

FIG. 14 illustrates a flowchart for the main parallel stage of analysis;

FIG. 15 illustrates the serial stage of analysis;

FIG. 16 illustrates a simple fight diagrammatic view of themicrofluidics chip;

FIG. 17 illustrates a simplified diagrammatic view of a parallel module;

FIG. 18 illustrates simplified diagrammatic view of a serial module;

FIG. 19 illustrates a simplified diagrammatic view of a serial modulearranged in parallel;

FIGS. 20A-20B illustrated a diagrammatic view of an embodiment utilizinga chemostat;

FIG. 21 illustrates a diagrammatic you have the microfluidics chiputilizing valves; and

FIGS. 22A-22B illustrate cross-sectional views of a micro valve.

DETAILED DESCRIPTION

Referring now to the drawings, wherein like reference numbers are usedherein to designate like elements throughout, the various views andembodiments of a microfluidic testing system with cell capture/analysisregions for processing a parallel and serial manner is illustrated anddescribed, and other possible embodiments are described. The figures arenot necessarily drawn to scale, and in some instances the drawings havebeen exaggerated and/or simplified in places for illustrative purposesonly. One of ordinary skill in the art will appreciate the many possibleapplications and variations based on the following examples of possibleembodiments.

Referring now to FIG. 1, there is illustrated a diagrammatic view of amicrofluidics chip 102 at a high-level view. There is provided in themicrofluidics chip 102 an input stage 104 that is operable to receive abiological specimen. As used herein, a “sample” must be capable offlowing through microfluidic channels of the system embodimentsdescribed hereinbelow. Thus, any sample consisting of a fluidsuspension, or any sample that be put into the form of a fluidsuspension, that can be driven through microfluidic channels can be usedin the systems and methods described herein. For example, a sample canbe obtained from an animal, water source, food, soil, air, etc. If asolid sample is obtained, such as a tissue sample or soil sample, thesolid sample can be liquefied or solubilized prior to subsequentintroduction into the system. If a gas sample is obtained, it may beliquefied or solubilized as well. The sample may also include a liquidas the particle. For example, the sample may consist of bubbles of oilor other kinds of liquids as the particles suspended in an aqueoussolution.

Any number of samples can be introduced into the system for analysis andtesting, and should not be limited to those samples described herein. Asample can generally include any suspensions, liquids, and/or fluidshaving at least one type of particle, cellular, droplet, or otherwise,disposed therein. In some embodiments, a sample can be derived from ananimal such as a mammal. In a preferred embodiment, the mammal can be ahuman. Exemplary fluid samples derived from an animal can include, butare not limited to, whole blood, sweat, tears, ear flow, sputum, bonemarrow suspension, lymph, urine, brain fluid, cerebrospinal fluid,saliva, mucous, vaginal fluid, ascites, milk, secretions of therespiratory, intestinal and genitourinary tracts, and amniotic fluid. Inother embodiments, exemplary samples can include fluids that areintroduced into a human body and then removed again for analysis,including all forms of lavage such as antiseptic, bronchoalveolar,gastric, peritoneal, cervical, athroscopic, ductal, nasal, and earlavages. Exemplary particles can include any particles contained withinthe fluids noted herein and can be both rigid and deformable. Inparticular, particles can include, but are not limited to, cells, aliveor fixed, such as adult red blood cells, fetal red blood cells,trophoblasts, fetal fibroblasts, white blood cells, epithelial cells,tumor cells, cancer cells, hematopoeitic stem cells, bacterial cells,mammalian cells, protists, plant cells, neutrophils, T lymphocytes,CD4+, B lymphocytes, monocytes, eosinophils, natural killers, basophils,dendritic cells, circulating endothelial, antigen specific T-cells, andfungal cells; beads; viruses; organelles; droplets; liposomes;nanoparticles; and/or molecular complexes. In some embodiments, one ormore particles such as cells, may stick, group, or clump together withina sample.

In some embodiments, a fluid sample obtained from an animal is directlyapplied to the system described herein at the input stage, while inother embodiments, the sample is pretreated or processed prior to beingdelivered to a system. For example, a fluid drawn from an animal can betreated with one or more reagents prior to delivery to the system or itcan be collected into a container that is preloaded with such a reagent.Exemplary reagents can include, but are not limited to, a stabilizingreagent, a preservative, a fixant, a lysing reagent, a diluent, ananti-apoptotic reagent, an anti-coagulation reagent, an anti-thromboticreagent, magnetic or electric property regulating reagents, a sizealtering reagent, a buffering reagent, an osmolality regulating reagent,a pH regulating reagent, and/or a cross-linking agent.

At this point in the process, a finite amount of biofluids is disposedin the reservoir ready for transferring to subsequent stages. Thisamount of fluid is then transferred to another stage via a driving stage106 in order to transfer this biofluid to another reservoir, thatassociated with a viewing stage 108. At this stage, a technician canexamine the biofluid and determine the makeup of the biofluid,discriminate cells, etc. in order to make certain decisions as to goingforward with remaining tests. The microfluidic chip then transfers thebiofluid at the viewing stage 108 to a parallel analysis stage 115through a parallel driving stage 110 wherein the biofluid is dividedamong a plurality of parallel path this for analysis of the reaction ofthe material in the biofluid with different reagents in a reading. Thisrequires a certain amount of the biofluid to be transferred to thisanalysis stage. Thereafter, a decision is made as to whether to transferthe remaining biofluid from the viewing stage 108, in order to performmore testing and/or analysis on the biofluid. At this stage the process,only one of the multiple second stage or serial stage path is selected.One reason for this is that there is only a finite amount of biofluidavailable and there is no need for testing along paths that areassociated with previous decisions indicating that the results will benegative along these paths. Each of these serial passes associated withone of the parallel paths. Thus, if there are five parallel paths, therewill be five serial paths. Note that the term “serial path” is a termmeaning that it is within the serial decision tree and it need notactually be a plurality of serial paths that are linked together in aserial manner, although they could be and are in some embodimentsdescribed hereinbelow. It is necessary to perform the testing/analysisalong each of the five parallel paths, but a decision at this pointindicates that only one of the serial paths will be required for thetesting/analysis purpose. This will be described in more detailhereinbelow.

Referring now to FIGS. 2A-2C, there are illustrated diagrammatic viewsof the various stages of the process. With specific reference to FIG.2A, there is illustrated a diagrammatic view of first viewing stage,wherein the amount of biofluid stored in the input stage reservoir 104is driven to the viewing stage 108 reservoir. At this stage, opticaldevice 202, for example, can be used to view the cells disposed withinthe medium. This medium could actually be the actual biofluid that wasprovided in the sample from the human/animal or could be some dilutedversion thereof. However, this biofluid will contain some cellularmaterial or some particulate of interest. This can be viewed with theout device 202 and then passed to a processor 204, or a human couldanalyze the results. With utilization of the processor 204, the actualform of biofluid, and analog form, is transferred to a digital form.This could be in the form of cell counting for verification of aparticular cell. As will be described hereinbelow, affinity labels canbe associated with each of the cells or particulates in the biofluid andthis could facilitate visual recognition of different characteristics ordifferent types of cells, such as proteins, bacteria, etc. Each of thesecellular materials can have a particular affinity label associated therewith that allows it to be visually identified via some characteristicssuch as florescence or even magnetic properties associated with theaffinity label. Again, this will be described hereinbelow. Although anoptical device 202 was illustrated and described, any other type ofdevice for analyzing the characteristics of a particular affinitylabeled cell can be utilized, such as some type of magnetometer, etc.

Referring now to FIG. 2B, there is illustrated the next parallel drivestage. At this stage, a micropump is utilized in the parallel drivestage 110 to pump at least a portion of the biofluid stored in thereservoir associated with the viewing stage 108 is transferred to all ofthe parallel reading/analysis paths. In this step, it can be seen that aportion of the biofluid in the reservoir associated with the viewingstage 108 and is biofluid exists in each of these parallel paths foranalysis. There is an indication in one of these parallel paths,associated with the reservoir 210, that shows a positive indication of areaction of some type that is viewable. If, for example, this werebacteria, one reagent could be an antibiotic in a large dosage thatwould destroy the particular target bacteria and this would berecognized by an observer. The other three paths, associated withreservoirs 214, 216 and 218 (an example of 4 paths), would have noreaction and, as such, would not have affected the bacteria associatedtherewith. In this example, a high level of concentrated antibiotic isprovided that would destroy the bacteria, but at this level of analysis,there is no indication provided as to the actual dosage of thatantibiotic that would destroy the bacteria, other than the fact that alarge dosage of this particular antibiotic will destroy the targetbacteria. It is important to keep in mind that this particular biofluidmay have multiple and different bacteria, proteins, etc. containedtherein.

Referring now to FIG. 2C, there is illustrated a diagrammatic view ofthe final serial stage of analysis/testing. Since the first stage oftesting/analysis transferred some of the biofluid from the viewing stage108 to the parallel stages 114, there is still some biofluid remainingin the viewing stage 108. This is a selectively transferred to one ofthe serial paths, that associated with the testing reservoir 210. Thereare provided a plurality of bypass channels 220 associated with each ofthe serial paths and only the bypass channel 220 associated with thereservoir 210 in the parallel path 114 will be selected for transferringbiofluid to this particular serial path associated with the reservoir210 for testing. It will first be pumped to be a micropump in a serialdrive stage 222 to a first serial reservoir 224 for testing/analysis. Ifthe test is negative, it can then be passed to a subsequent serialdriving stage 226 to a subsequent serial reservoir 228 fortesting/analysis and so on. As will be described hereinbelow, there canbe provided a single bypass path 220 which is connected to a manifoldassociated with each of the serial paths and each of the manifolds canbe associated with each of the different reservoirs for testing, i.e.,at this point the testing is parallel to all of the subsequent testingreservoirs. In the mode illustrated in this FIG. 2C, it is necessary totransfer all of the necessary biofluid, i.e., typically the remainingbiofluid in the viewing stage reservoir 108, to the reservoir 224 andpass all of that biofluid to the next reservoir 228 and so on. Thus, ateach stage, all of the biofluid transferred in the subsequent stages istested at each subsequent stage. In a parallel configuration, theremaining biofluid in the viewing stage 108 would be required to bedivided among the different testing reservoirs at each of the subsequentstages. This will be described in more detail hereinbelow.

Referring now to FIGS. 3A-3D, there are illustrated diagrammatic viewsof the process and fluid flow. In FIG. 3A come there is illustrated anoverall process flow for the embodiment described hereinabove. Thisembodiment, there is provided an input well 302 for receiving thebiologic sample indicated by numeral 303. This constitutes a finitevolume that must be transferred via a micropump to a viewing reservoir306. At this point, substantially all of the biofluid is transferredfrom the reservoir 302 to the viewing reservoir 306. This is the firststage of the process. The second stage of the process is illustrated asproviding three separate testing reservoirs 308, 310, 312, attached atone to a microchannel manifold 314. Each of the testing reservoirs 308,310, 312, as will be described hereinbelow, is comprised of a serpentinemicrochannel 316 attached at one end to the manifold 314 and at theother end to a viewing reservoir 318. A micropump 320 is provided fortransferring biofluid from the viewing reservoir 306 to the manifold314. This will be divided among the three testing reservoirs 308, 310,312 and substantially even amounts. The biofluid will traverse theserpentine microchannel 316, which is coated with a particular reagent,one example being an antibiotic. In this example, the antibiotic is at avery high concentrated level, each of the testing reservoirs 308, 310and 312 having a different antibiotic associated there with. Only aportion of the biofluid in the viewing reservoir 306 will be transferredto these three testing reservoirs 308, 310 and 312 for testing/analysisand viewing at the associated viewing reservoir 318. The serpentineshape, when used with a medium containing cells such as in a biologicsample, facilitates and enhances mixing due to the increased interfacialcontact area between the cells within the biofluid sample.

The next step of testing/analysis will be selected only upon a positivetest occurring within one of the three testing reservoirs 308, 310 and312. However, each of the testing reservoirs 308, 310 and 312 hasassociated there with a subsequent group of testing reservoirs. In thisembodiment, each of the subsequent testing reservoirs is comprised of aplurality of sub reservoirs 330, each of the sub reservoirs 330 beingconfigured identical to the testing reservoirs 308, 310 and 312, with aserpentine microchannel region 316 and a viewing reservoir 318. A singlebypass microchannel 220 is provided to connect viewing reservoir 306 toa sub reservoir manifold 332. Each of the particular sub reservoir pathshave associated there with a separate micropump 334. Only one of thesemicropumps 334 is selected for transferring the remaining portion of thebiofluid stored in the viewing reservoir 306 to the selected path. Inthis embodiment, the remaining portion of the biofluid is transferred tothe first reservoir 330 bypassing the biofluid through the serpentinemicrochannel 316 to the associated viewing reservoir 318. Thisparticular microchannel will have coating of antibiotic, in this exampleabove, at a relatively low dose. If the bacteria, for example, do notreact accordingly with this level of antibiotic, it can be recognized assuch in the viewing reservoir 318. It is noted that the antibioticassociated with the coating on the walls of the microchannel 330 at thisdosage will not be picked up by the bacteria and, as such, the bacteriain the viewing reservoir 318 for the first sub reservoir 330 in theselected path will still be intact. It can then be pumped from thereservoir 318 associated with the first testing reservoir 330 in thechain to a subsequent testing reservoir 330 with a subsequent micropump336. This subsequent sub reservoir will have a concentration ofantibiotic in its serpentine microchannel 316 that is at a higher level.As the level increases, a gradient is tested for, such that the dosagecan be gradually increased until the bacteria are destroyed. If, forexample, the bacteria were associated with an affinity label that madeit fluoresce, this would be recognized. It could also be that there aremultiple bacterial types contained within the biofluid that are eachassociated with a different affinity label and this could be recognized.It could, in fact, the case that one type of bacteria perfected at afirst dosage level of the antibiotic and a second bacteria were affectedat a another dosage level of the antibiotic.

Referring now to FIG. 3B, there is illustrated a diagrammatic view of analternate process flow. This will work substantially identical to theembodiment of FIG. 3A, come up until the operation at the manifold 332associated with the sub reservoirs. In this embodiment, the threemicropumps 334 each feed a sub reservoir manifold 340. Each of the subreservoir manifolds 340 is connected to a plurality of the subreservoirs 330 associated with each path. In this embodiment, there areonly illustrated three sub reservoirs 330 for each of the sub reservoirmanifolds 340, although each path could have a different number of subreservoirs 330 associated therewith. The difference between these twoembodiments is that, at this point, the amount of biofluid remaining inthe viewing reservoir 306 now must be divided amongst all of the subreservoirs attached on one end thereof to the associated sub reservoirmanifold 340 selected by the activated one of the micropumps 334. Thiswill result in potentially less biofluid being available for thetesting/analysis step. This will also mean that each of the viewingreservoirs 318 associated there with will have a smaller volumeassociated therewith.

Referring now to FIG. 3C, there is illustrated a diagrammatic view thatprovides a simplified diagram of the transfer from reservoir toreservoir. In this illustration, the input stage is illustrated as aninput reservoir 350 labeled R0. A micropump 352 is operable to transferthe contents of this input reservoir, the biofluid, to a secondreservoir, a viewing reservoir 354, labeled R1. A portion of thecontents of this reservoir are then transferred via a micropump 356 to aplurality of parallel stage reservoirs 358 labeled R2. This is the firsttesting/analysis stage. After this stage, the remaining contents of theviewing reservoir 354 are transferred to the subsequent serial stagereservoirs via a pump 360 via a bypass path and microchannel 362. Theserial stage reservoirs are labeled R3, R4, etc. This illustration setsforth how the entire contents of the input reservoir R0 are transferreddown the chain. This is best illustrated in FIG. 3D. In thisillustration, it can be seen that entire contents of reservoir R0 aretransferred to reservoir R1. At this point, only a portion of thecontents are transferred to reservoir R2. The remaining contents aresequentially transferred to R3, R4, and so on. For this illustration,the entire remaining contents of the reservoir 354, R1, will betransferred down the chain entirely to reservoir R3, then to reservoirR4, and so on. In the alternate embodiment, as described hereinabove,and not illustrated in FIG. 3D, the bypass 362 could be connected toeach of the reservoirs R3, R4, etc. in parallel, noting that theremaining contents of the reservoir R1 will then be divided amongst theparallel connected reservoirs R3, R4, etc.

Referring now to FIGS. 4A-4G, there are illustrated diagrammatic viewsof the initial processing section associated with the viewing stage 108.There is provided a substrate 402 upon the surface of which are formed aplurality of wells and microchannels. A first well 404 is provided forreceiving the biofluid sample in this well has a finite volumeassociated there with. At the bottom of this well a microchannel 406extends outward and up to the surface to an opening 408. The purpose ofthis microchannel 406 extending to the bottom of the well 404 is toensure that the biofluid can be completely pumped from the well 404. Forthe formation of this microchannel 406, it might be that themicrochannel is formed through the surface of the substrate 402 and thena cover plate (not shown) having a surface that extends down into theopen microchannel. An adjacent channel 410 is disposed proximate theopening 408 to provide another opening therefore in order to accommodatea micropump 412 (shown in phantom) interface with the opening 408 andthe one end of the microchannel 410 for transferring fluid from the well404 to the microchannel 410. The microchannel 410 extends along thesurface of substrate 402 in order to interface with a viewingwell/reservoir 412. As the biofluid passes through the microchannel 410and the viewing well 412, a desired analysis can be performed on thecontents of the biofluid. As described hereinabove, in one example,various cells in the biofluid might consist of different types ofbacteria, proteins, etc. and each of these may have associated therewith a specific affinity label, which is optically detectable. Thereare, of course, other means by which affinity labels can be detected. Asthe cells contained within the biofluid pass through the viewingwell/reservoir 414, they can be examined. The viewing well/reservoir 414on the other side thereof is connected to one side of a microchannel416, the other side thereof connected to a reservoir 418. Since themicropump 412 must force the biofluid through the microchannels and theviewing well/reservoir 414, there is required the necessity for aholding reservoir 418 to be present. However, initially, this reservoir418, the microchannel 410 and the viewing well/reservoir 414 will haveair disposed therein. This air must be removed. This can be done with anegative pressure of some sort or just a waste gate output to theatmosphere. This is provided by a waste gate microchannel 420 that isconnected to an opening 422 through the cover glass (not shown) or tothe side of substrate 402. A valve 423 could be provided above theopening 422. As biofluid enters the reservoir 418, air will be pushedout through the microchannel 420. It is desirable for this microchannel422 to have as low a profile as necessary such that only air exitstherefrom. Depending upon the size of the cells contained within thebiofluid, the microchannel 420 can be significantly smaller and have alower profile than the microchannels 410 and 416. Is important to notethat, once the micropump 412 transfers the biofluid from the well 404,the volume transferred will be spread between the two microchannels 410and 416, the viewing well 414 and the reservoir 418. Thus, the reservoir418 has a significantly larger volume that any of the microchannels 410and 416 and the viewing well/reservoir 414. Additionally, it may be thatthe depth of the wells/reservoirs 404 and 418, as well as the viewingwell reservoir 414 are also as shallow as the microchannels 410 and 416but significantly wider to accommodate the required volume.

The outlet of the reservoir 418 is connected from the bottom thereofthrough a microchannel 426 to an opening 428 on the upper surface of thesubstrate 402. This is interfaced with a micropump 430 (in phantom) toan adjacent microchannel 432 for subsequent processing. These micropumps412 and 430, although illustrated as being flush with the substrate,will typically be disposed above the cover plate (not shown) with holesdisposed through the cover plate. The opening 428 will be a horizontalmicrochannel associated with the manifold 314 described hereinabove.This will be associated with a plurality of micropumps 430 for each ofthe parallel paths or the bypass path. A cross-sectional view of theembodiment of FIG. 4A is illustrated in FIG. 4B, with a cover plate 440disposed over the substrate 402 with an opening 442 disposed above thewell 404 for receiving the biofluid sample.

FIGS. 4C and 4D illustrate top view and cross-sectional views of thereservoir 418 illustrating how the microchannel 416 feeds biofluid intothe top of the reservoir 418, and the flow path for the biofluid fromthe reservoir 418 through the microchannel 426 from the bottom of thereservoir 418. However, it may be that, with capillary action, the depthof the reservoir 418 could be equal to that of the microchannels 416 and426 such that they are all at the surface of the substrate 402 for easeof manufacturing. When a negative pressure is placed upon the reservoir418, air will be pulled into the microchannel 426 through themicrochannel 420. It is possible in this mode that the micropump 412could be operated to actually create a positive pressure in themicrochannel 416 to force the biofluid in the reservoir 418 into theopening 428 through the microchannel 426. Again, the microchannel 420would preferably have a dimension that was smaller than the smallestcell size within the biofluid.

Referring now to FIGS. 4E and 4F, there are illustrated top view andcross-sectional views of the reservoir 418 with an alternate embodimentillustrating microchannel 426′ as being beneath the bottom of thereservoir 418 to allow more complete emptying of the reservoir 418.

Referring now to FIG. 4G, there is illustrated an alternate embodimentof inlet wells for receiving the biofluid sample. There is provided thewell 404 for receiving the biofluid sample and a second well 464receiving an additional fluid sample. This fluid sample in well 460could be some type of dilutant or it could be a medium containingvarious affinity labels. As noted hereinabove, the fluid sample couldhave associated there with affinity labels prior to the biofluid samplebeing disposed in the well 404. However, it is possible that themicrofluidic chip have disposed in the well 460 a medium containingaffinity labels, for example. The well 460 would be interfaced through amicrochannel 462 to an opening 464 adjacent the opening 408. A twoinput, one output, micropump 412′ that interfaces with the microchannel410.

Referring now to FIG. 5, there is illustrated a diagrammatic view of themicrochannel structure associated with the parallel stage of operation.The microchannel 426 is interfaced with a microchannel manifold 502which corresponds to the opening 428. This microchannel manifold 502 isinterfaced with a plurality of micropumps 504, corresponding to themicropump 430. These micropumps 504 are disposed in pairs, each pairassociated with one testing reagent. As noted hereinabove, there areprovided a plurality of parallel paths, each associated with a reservoir312 having a serpentine microchannel 316 and a viewing reservoir 318.The first micropump 504 in the pair of micropumps 504 is connected toone end of the associated serpentine microchannel 316. When thismicropump 504 is activated, biofluid from the reservoir 418 is passedthrough the manifold microchannel 502 and through the serpentinemicrochannel 316 to the viewing reservoir 318. As was the case above,there is provided a waste microchannel 506 for each of the reservoirs318 to allow air to escape therefrom as biofluid is forced through themicrochannel 316. The micropump 504 associated with this serpentinemicrochannel 316 will be operated for a sufficient amount of time totransfer sufficient biofluid from the reservoir 418 through theserpentine a channel 316 and finally into the reservoir 318 to fill thereservoir 318. The microchannel 506 can have some type of valveassociated with the opening thereof to prevent the escape of anybiofluid therefrom or, alternatively, the dimensions of thatmicrochannel 506 could be small enough to prevent any appreciable amountof cells escaping therefrom. Although not illustrated, the one of thepair of micropumps 504 associated with the parallel stage of operationand associated reservoirs 312 will also be operated to fill theassociated serpentine microchannel 316 and reservoir 318.

Referring now to FIGS. 5A and 5B, there are illustrated cross-sectionalviews of the serpentine microchannel 316. As described hereinabove, thesides of these channels 316 are coated with some type of reagent. Forexample, if a Urinary Tract Infection (UTI) were suspected and werebeing tested for in the microfluidic chip, the sensitivity for commonantimicrobial agents for UTI treatment might include ampicillin (AMP),ciprofloxacin (CIP), and trimethoprim/sulfamethoxazole (SXT), thesebeing three agents that could be tested for and three different paths.The bacteria that might exist within the urine samples from anindividual could be any of uropathogenic E. coli strains (EC132, EC136,EC137, and EC462). Some prior research has shown that, throughantimicrobial resistance profiles of these pathogens that EC132 isresistant to AMP and CIP but not SXT. EC136 is resistant to AMP only.EC137 is sensitive to all the antibiotics tested. EC462 is resistant toAMP and SXT but not CIP. In order to coat sides of the serpentinemicrochannels 316, one technique would to have a certain amount of theantibiotic dissolved in sterile water to the serpentine microchannels316 at different levels. Subsequently, the diluted antibiotic is driedby incubation at a desired temperature and desired time. The originaldiluted antibiotic has a starting concentration of a predetermined μg/mlconcentration. The surface area is sufficiently covered such that, whenthe biofluid passes thereover, it will interact with reagent.

Referring now to FIG. 6, there is illustrated a microchannel diagram ofthe reservoir 330 on the surface of the chip 402. This is connected bythe microchannel 507 from the associated one of the micropumps 504.After the results in the viewing reservoir 318 have been determined toyield a positive result, for that particular path in the parallelanalysis/testing operation, the other of the pair of micropumps 504 isactivated and the remaining amount of micro-fluid from the reservoir 418is transferred to the reservoir 330. This will be passed through theserpentine microchannel 316 and stored in the reservoir 318, labeled 602in FIG. 6. This is substantially larger than the reservoir 318associated with the reservoir 312. Thus, for this embodiment, theremaining portion of the biofluid from the reservoir 418 will besubstantially stored in the reservoir 602. This will have associatedthere with a waste microchannel 604 and an outlet microchannel 608 thatextends outward from the bottom of the reservoir 602 and up to anopening 610 in the surface of the substrate for interface with themicropump 336. The micropump 336 is operable, at the next stage of thetesting/analysis, to move the contents of the reservoir 602 over to thenext reservoir 330 for testing at that next concentration levelassociated with the next reservoir 330 in the sequence.

Referring now to FIGS. 7A-7D, there is illustrated an example of avalveless MEMS micropump. The micropump includes a body 702 with twopumping chambers 704 and 706. At the inlet side of each of the chamber704 and 706 is disposed a conical inlet 710 and 712, respectively. Theconical inlets 710 and 712 are wider at the pump chamber side andnarrower at the inlet side thereof. The inlet sides of conical inlet 710and 712 are connected to respective inlet channel 714 and 716. Theoutlet side of the chambers 704 and 706 are interfaced with conicaloutlets 718 and 720, respectively, the conical outlets 718 and 720having a narrower portion at the outlet of the respective pump chamber704 and 706 and a wider portion at the respective outlet thereofinterfacing with respective outlet channels 722 and 724. The conicalinlets 710 and 712 and outlets 718 and 720 are frustro conical in shape.A piezoelectric membrane and actuator 726 is dispose between the twopumping chambers 704 and 706 and is operable to be extended up into oneof the chambers 704 and 706 at one time to increase the pressure thereinand at the same time extend away from the other of the chambers 704 and706 to decrease the pressure therein. The operation is then reversed.

The piezoelectric membrane and actuator 726 is comprised of apiezoelectric disc 740 on one side of a membrane 742 and a piezoelectricdisc 744 on the other side thereof. Each of the piezoelectric discs 740and 744 are formed by stratifying a layer of use electric material 748between two layers of conducting material 750. Piezoelectric material748 can be made with Piezo Material Lead Zirconate Titanate (PZT-SA),although other piezoelectric materials can be used. The conductingmaterial 60 may be composed of an epoxy such as commercially availableEPO-TEK H31 epoxy. The epoxy serves as a glue and a conductor totransmit power to the piezoelectric discs 750. The piezoelectric discs750 are secured to the surface of the intermediate layer 748, so thatwhen a voltage is applied to the membrane 742, a moment is formed tocause the membrane 742 to deform.

The operation of the micropump will be described with reference to FIG.7D. At rest, the upper chamber 704 and the lower chamber 706 areseparated by a diaphragm pump membrane 742. The diffuser elements 710,712, 718 and 720 are in fluid communication with each respectivechamber. Diffuser elements are oriented so that the largercross-sectional area end of one diffuser element is opposite the smallercross-sectional area end of the diffuser element on the other side ofthe chamber. This permits a net pumping action across the chamber whenthe membrane is deformed.

The piezoelectric discs are attached to both the bottom and the top ofthe membrane. Piezoelectric deformation of the plates is varied byvarying the applied voltage so as to excite the membrane with differentfrequency modes. Piezoelectric deformation of the cooperating platesputs the membrane into motion. Adjustments are made to the appliedvoltage and, if necessary, the choice of piezoelectric material, so asto optimize the rate of membrane actuation as well as the flow rate.Application of an electrical voltage induces a mechanical stress withinthe piezoelectric material in the pump membrane 742 in a known manner.The deformation of the pump membrane 742 changes the internal volume ofupper chamber 704 and lower chamber 706. As the volume of the upperchamber 704 decreases, pressure increases in the upper chamber 706relative to the rest state. During this contraction mode, theoverpressure in the chamber causes fluid to flow out the upper chamber704 through diffuser elements on both sides of the chamber. However,owing to the geometry of the tapered diffuser elements, specifically thesmaller cross-sectional area in the chamber end of the left diffuserelement relative to the larger cross-sectional area of the rightdiffuser element, fluid flow out of the left diffuser element is greaterthan the fluid flow out the right diffuser element. This disparityresults in a net pumping of fluid flowing out of the chamber to theleft.

At the same time, the volume of the lower chamber 706 increases with thedeformation of the pump member 742, resulting in an under pressure inthe lower chamber 706 relative to the rest state. During this expansionmode, fluid enters the lower chamber 706 from both the left and theright diffuser elements. Again owing to the relative cross-sectionalgeometry of the tapered diffuser elements, fluid flow into the lowerchamber 706 through the right diffuser element is greater than the fluiddrawn into the lower chamber 706 through the left diffuser element. Thisresults in a net fluid flow through the right diffuser element into thechamber, priming the chamber for the pump cycle.

Deflection of the membrane 742 in the opposite direction produces theopposite response for each chamber. The volume of the upper chamber 704is increased. Now in expansion mode, fluid flows into the chamber fromboth the left and right sides, but the fluid flow from the rightdiffuser element is greater than the fluid flow from the left diffuserelement. This results in a net intake of fluid from the right diffuserelement, priming the upper chamber 704 for the pump cycle. Conversely,the lower chamber 706 is now in contraction mode, expelling a greaterfluid flow from the lower chamber 706 through the left diffuser elementthan the right diffuser element. The result is a net fluid flow out ofthe lower chamber 706 to the left.

Referring now to FIG. 8, there is illustrated a cross-sectional view ofa piezoelectric micropump with check valves. Membrane 802 is disposedwithin a pump chamber 804 and secured to a body 806. A piezoelectricdisc 808 is disposed beneath the membrane 802 and electrode 810 isdisposed below the piezoelectric disc 808. Deformation of the membrane802 with the piezoelectric disc at the appropriate frequency will causea volume of the pumping chamber 804 to change. An inlet valve 811 allowsfluid to flow into the chamber 804 and an outlet valve 812 allows fluidto flow out of the chamber 804.

Referring now to FIG. 9, there is illustrated a micropump 960 in which ananofabricated or microfabricated fluid flow pathway is formed betweenstructures. A first reservoir 961 terminates with a first gate valve 966which permits or restricts fluid flow between the first reservoir 961and a second reservoir 971. An electrolytic pump 985 drives a firstdiaphragm 965 which is communication with the second reservoir 971, toclose the first gate valve 966, and pulls a second diaphragm 969, whichopens a second gate valve 968 to drive fluid from the second reservoir971 to a third reservoir 973. The electrolytic pump 985 is driven byelectrowetting of a first membrane 964 on the first gate valve 966 sideof the pump. By switching to electrowetting of a second membrane 963fluid within the third reservoir 973 is emitted from an exit opening 970by actuation of the second diaphragm 969.

Referring now to FIG. 10, there is illustrated a flowchart depicting theoverall operation of the system. The process is initiated at a Startblock 1002 and then proceeds to a block 1004, wherein the biofluidsample is loaded. The process enclosed a block 1006, wherein thebiofluid is transferred to the viewing window or the cell counter. Theprocess then flows to a decision block 1008 to determine when thecounting operation is done, i.e., when the cells have beendiscriminated. As noted hereinabove, each of these cells could beassociated with, depending on upon the type, a particular affinity labelto allow them to be discriminated between within the viewing window. Theprocess then flows to a block 1010 in order to pump the biofluidmaterial to the next phase, that associated with the paralleltesting/analysis step. A decision is then made at a block 1012 as towhether this is a positive state, i.e., has any of the biofluid materialinteracted with a particular reagent to give a positive result. If not,the process is terminated at a block 1014 and, if so, the process flowsto a block 1016 in order to capture the biofluid material in a secondaryreservoir. Once the path is selected, the appropriate micropump isactivated and the biofluid material is pumped to the next reservoiralong the secondary path, as indicated by a block 1018. The process thenflows to a block 1020 in order to analyze the results at each secondaryreservoir and, if there is a positive result, as indicated by block1022, the process is terminated at a block 1024. If the result is notpositive, the process then flows to a block 1026 to determine if that isthe last testing reservoir and, if so, the process flows to theterminate block 1024. If there are more testing/analysis blocks throughwhich to process the biofluid material, the process then flows to block1028 and back to the input of a block 1018 to pump the biofluid serialto the next testing reservoir.

Referring now to FIG. 11, there is illustrated a flowchart for theloading operation, which is initiated at a block 1101 and then flows toa block 1102 wherein the sample is placed in the well and then to adecision block 1104 to determine if this is a process wherein thebiofluid sample is to be mixed with some other diluted product or anaffinity label. If it is to be mixed, the process flows to a block 1106to mix the biofluid sample and, if not, the process bypasses this step.The process then flows to a block 1108 in order to activate the pump andtransferred the biofluid material after mixing to the next reservoir inthe process.

Referring now to FIG. 12, there is illustrated a flowchart for theprocess of the cell counting operation, i.e., the operation at theviewing reservoir. This is initiated at a block 1202 proceeds to a block1204 in order to transfer the biofluid material to the viewing chamber.The process enclosed a block 1206 in order to view the cells in realtime as they pass through the various microchannels and viewing window.The process then flows to a block 1208 in order to count the cells. Atthis stage, the cells can have various affinity labels associated therewith such that the target cells can be viewed and discriminated betweenbased upon the affinity labels associated therewith. If, for example,there were multiple types of bacteria contained within the biofluidsample and each of these types of bacteria had associated therewithdifferent affinity label that clips arrest at a different color, theykilled be discriminated between. Additionally, proteins would have adifferent affinity label than a bacteria and this would also allowdiscrimination between the two types of cells. The process then flows toa block 1210 to store the transferred biofluid in the reservoir and intoa block 1212 to terminate.

Referring now to FIGS. 13A-13C from their illustrated variousconfigurations for the cell counting operation. In the first embodimentof FIG. 13A, there are provided a three-part microchannel 1302, a middlesection microchannel 1304 and an outlet microchannel section 1306 themiddle section 1304 has a diameter that is slightly larger than thelargest cell that could be contained within the biofluid. This allowsthe cells to be transferred in a more orderly manner. The cell viewingwould be performed at this middle section microchannel 1304. In theembodiment of FIG. 13B, there are provided three varying diameter middlemicrochannel sections 1308, 1310 and 1312, each with different diametersto allow different size cells to flow therethrough. This type ofembodiment may facilitate some selection in the cells for viewing. Inthe embodiment of FIG. 13C, there is illustrated the above discloseembodiment wherein the microchannel 416 empties into the reservoir 418and the viewing is basically performed upon the cells within thereservoir 418.

Referring now to FIG. 14 come there is illustrated a flowchart for theparallel cell capture in the first testing/analysis stage. This isinitiated at a block 1402 and a process and proceeds to a block 1406 inorder to preload all of the cell capture areas having reagent associatedthere with, such that the portion of the biofluid stored in thereservoir 418 is transferred to the reservoirs associated with theparallel cell capture areas. The process enclosed a block 1408 whereinthe pump is activated to fill all of the cell capture wells associatedwith this stage of testing/analysis. The process then flows to a block1410 to possibly allow the cells to slowly go through the microchannelsin order to interact with the reagent. If so, this requires a certainamount of time and this would result in the micropumps operating at alower rate to allow sufficient time for the cells to flow through theserpentine microchannels 316 to interface with the particular coating onthe surfaces thereof. This basically is the amount of time required forthe micropumps to fill up the reservoir 318 associated there with. Thelength of the serpentine microchannel 316 would determine the amount oftime required to fill up the reservoir 318. Once the reservoir has beenfilled, as indicated by a block 1412, then the viewing window in thereservoir 318 is analyzed, as indicated by a block 1414. The path fromthe block 1410 to the input of the block 1414 indicates a path by whichthe micropumps can be run at a higher rate. The process then isterminated at a block 1416.

Referring now to FIG. 15, there is illustrated flowchart for the secondphase of the analysis, provided that the first phase indicated apositive result for one of the cell capture areas and the associatedreagent. This is initiated a block 1502 and then proceeds to a block1504 to preload all of the secondary cell capture areas with reagent andinto a function block 1506 to pump all of the remaining biofluidmaterial from the reservoir 418 into the first reservoir in thesecondary reservoirs 330. This also goes through and incubate step toallow the micropumps to pump at a slower rate to allow the biofluidmaterial to go through the serpentine microchannel 316 at a slower ratebefore it enters the associated reservoir 318. When the reservoir 318 isfilled, as indicate a by block 1510, the contents of the reservoir 318are analyzed at a block 1512. If the pump can be run at a faster rate,this is provided by a path around the block 1510. If the result ispositive, as indicated by a block 1514, then the process is terminatedat a block 1516. If not, the process flows from the block 1514 to ablock 1518 in order to the next reservoir 330 in the back to the inputof the serpentine microchannel 316 and then float the input of the block1508.

Referring now to FIG. 16, there is illustrated a simplified diagrammaticview of the microfluidics chip for processing a plurality of modules.The sample 303 is input to the well 302 and then pumped into the viewingwindow 306. A waste microchannel 1602 is provided an interface to theviewing window 306 that is interfaced with a micro valve 1604 to allowair to escape, or any bubbles that may be present, from the viewingwindow 306. Additionally, the waste microchannel 1602 could interfacewith an external vacuum source aid in fluid flow. A cellcounter/discriminator 1606 is provided for optically viewing thecontents of the viewing window 306, the output thereof processed via aprocessor 1608. The outlet of the viewing window 306 is interfaced witha manifold microchannel 1610 through a connecting channel 1612. At thispoint, the micro valve 1604 is closed such that the biofluid containedwithin the viewing window 306 and the interfaced with microchannelmanifold 1610 to allow fluid to be pump therefrom to a plurality ofdistribution paths along distribution microchannels 1614. It may be thatpump 304 would need to be activated in order to reduce the pressure atthe upper end of the viewing channel 306 or, alternately, a microchannel1618 interfaced with a micro valve 1620 could be provided to, when open,relieve the pressure in the upper end of the viewing window 306 to allowbiofluid to be pumped therefrom to the microchannel manifold 1610.

Each of the distribution microchannels 1614 is interfaced with aseparate module via an associated distribution pump 1624 to interfacewith and associated one of modules 1625, labeled A-Z, for example. Therecan be any number of modules provided. However, each module 1625 hasassociated there with a finite capacity and, therefore, the number ofmodules 1625 that can be interfaced to the viewing window 306 is afunction of the volume of biofluid contained therein and the capacity ofthe reservoirs of each of the individual modules 1625, each of theindividual modules 1625 potentially having a different capacity,depending upon the configuration thereof. However, selecting among thevarious distribution pump 1624 can allow desired tests to be done withthe available biofluid contained within the viewing window 306.

Referring now to FIG. 17 there is illustrated a diagrammatic view of oneof the modules 1625 associated with the parallel testing configuration,wherein biofluid is loaded into a plurality of testing reservoirs. Thedistribution pump 1624 associated there with transfers fluid from thedistribution microchannels 1614 to an intermediate microchannel manifold1702 which is then interface with a plurality of testing reservoirs 312,as described hereinabove. Each of these testing reservoirs has aserpentine microchannel 316 and a viewing window 318 associated therewith. As described hereinabove, each of these testing reservoirs canhave a different volume and a different configuration mechanically andcan be associated with a different test. They can each have a particularcoating of reagent, such as an antibiotic, to interact with the biofluidfor testing purposes to determine if there is any reaction of thebiofluid in the cells contained therein to the material coated on thesides of the serpentine microchannels 316. In the operation of thisparticular module 1625, all of these testing reservoirs 312 areassociated with different reagents and will be loaded in parallel. Forthis embodiment, will be desirable for each of the reservoir 312 to havethe same volume. If, however, they had different volumetric capacities,it would be necessary to have some type of waste gate with a micro valveto allow all of the viewing windows 318 to achieve full capacity.

Referring now to FIG. 18, there is illustrated a diagrammatic view ofthe serial wherein a plurality of testing reservoirs 330 is arranged ina series configuration. In this configuration, the associateddistribution pump 1624 will transfer biofluid from the microchannelmanifold 1610 through the distribution microchannels 1614 to the firstof the testing reservoirs 330. The biofluid will be contained within theviewing chamber 318 and, as noted hereinabove, there will be possible hesome type of waste microchannel associated micro valve to allowair/bubbles to escape during filling of the viewing window 318.Thereafter, a second serial pump 1706 is activated to transfer thecontents of the viewing window 318 to a second testing reservoir 330 inthe associated serpentine microchannel 316 and viewing window threeeight teen. In this transfer, there may be required a reliefmicrochannel (not shown) at the inlet end thereof to reduce the pressuretherein during the pumping operation. This will continue until all ofthe tests have been done. Each of the serpentine microchannels 316associated with each of the testing reservoirs 330 will have a graduatedincrease in the particular reagent to determine the dosage, in thisexample. It may be that, upon being exposed to the dosage of the reagentin the first testing reservoir 330 that cellular material in thebiofluid is somewhat affected by the reagent, i.e., the antibiotic, forexample. By moving to a higher concentration of the reagent in the nextsequential testing reservoir 330, this could be accounted for in theoverall analysis. It may be that the actual concentration in the nextsequential testing chamber 330 is not an exact incremental increase inthe reagent. For example, if it was desired to expose the biofluid toreagent increments of 10%, 20%, 30%, etc. in 10% increments, it may bethat the first testing chamber 330 has a concentration of 10% and thenthe second testing chamber has a concentration of possibly 16%,accounting for the fact that the accumulated effect of passing throughthe 10% testing chamber 330 and the 16% testing chamber 330 effectivelyprovides a 20% accumulated exposure in the second testing chamber 330and so on.

Referring now to FIG. 19, there is illustrated a diagrammatic view of aconfiguration for providing parallel loading of the serial configurationfor the incremental testing. This is similar to the embodiment of FIG.17, except that the testing chambers 330 are all interfaced with theassociated distribution pump 1624 through a microchannel manifold 1902in a parallel configuration, such that they are all loaded at the sametime, with each having a different concentration of reagent associatedthere with. In this configuration, however, since all of the testingchambers 330 will be loaded in parallel, there are required to be asufficient volume of biofluid contained within the viewing window 306initially to facilitate complete filling of each of the associatedviewing windows 318.

Referring now to FIGS. 20A-20B come there is illustrated a diagrammaticview of chemostat, wherein the associated distribution pump 1624transfers biofluid from the distribution microchannel 1614 two eightchemostat 2002. The details of the chemostat 2002 are illustrated inFIG. 20B. A main microchannel 2004 is interfaced on one and thereof withthe output of the distribution pump 1624 associated there with, with theother end of the microchannel 2004 interfaced with a waste gate via amicro valve (not shown). There are a plurality of cell storagemicrochannels 2006 connected between one surface of the mainmicrochannel 2004 and a waste microchannel 2008. Each of these cellstorage microchannels 2006 associated there with a filter 2010 disposedat the end thereof proximate to the waste microchannel 2008. Each of thecell storage microchannels 2006 has a size that will receive aparticular target cell having a particular dimension, such that thetarget cell will flow into the cell storage microchannel and cells ofsmaller size will pass through the associated filter 2010, which filter2010 is a microchannel with a diameter that is smaller than that of thetarget cell. This waste material will flow out through the waste gate ormicro valve (not shown) associated with the waste microchannel 2008. Bymaintaining a pressure differential between the main microchannel 2004and the waste microchannel 2008, the target cells will be stored withinthe cell storage channels 2006. Larger cells than the target cells inthe main microchannel 2004 will bypass the cell storage microchannels2006 and pass out of the waste gate associated with the mainmicrochannel 2004, keeping in mind that there is required to be a lowerpressure within the waste microchannel 2008 as compared to the mainmicrochannel 2004.

Referring now to FIG. 21, there is illustrated an embodiment of themicrofluidic chip utilizing micro valves as opposed to intermediatemicropumps. In this embodiment, there are illustrated a plurality ofinput wells 2102 for interfacing with an initial micropump 2104 to pumpfluid through a viewing window 2106 to a first reservoir 2108. Havingmultiple wells 2102 allows multiple samples to be input through theviewing window 2106 or to actually mix two different materials togetherfor flowing through the viewing window 2106 to the reservoir 2108. Thewaste gate 2110 can be provided at the reservoir connected thereto via awaste microchannel 2112 to allow air/bubbles to escape. A micropump 2114is operable to pump fluid from the reservoir 2108 to a main microchannelmanifold 2116. During this pumping operation, some type of pressurerelief is required which can either be provided via one of the pumps2104 being activated or a relief micro valve 2118 Interface with theinput end of the viewing window 2106 through a relief microchannel 2120.

Interfaced with the main microchannel manifold 2116 is a plurality ofdistribution micro valves 2124. These distribution micro valves 2124 canbe interfaced with various modules, as described above herein withrespect to FIGS. 17-20A/B. The only difference is that the associateddistribution pump 1624 has been replaced by a distribution valve 2124.Additionally, each of the parallel loaded testing reservoirs 312 can beindividually associated with one of the distribution valves 2124 toselectively certain ones thereof for testing. Since each one of thesetesting reservoirs 312, after selection, is required to be completelyfilled, by allowing individual selection of the testing reservoirs 312,certain ones thereof can be eliminated. It may be that, in pre-analyzingthe biofluid sample, it can be predetermined that certain ones of theassociated reagents in the reservoir 312 are not required thetesting/analysis step and can therefore be eliminated from the step offilling. This is opposed to the embodiment of FIG. 17, wherein all ofthe testing reservoirs 312 are complete the filled.

Referring now to FIGS. 22A-22B, there is illustrated cross-sectionalviews of a micro valve in an open and a closed position. The substrate402 has cover plate 440 disposed on top thereof. There are provided tomicrochannels 2202 and 2204 that are to be connected together with themicro valve. The microchannel 2202 is interfaced with a hole 2006 to thesurface of the cover plate 440 to an opening 2208. The microchannel 2204is interfaced to a hole 2210 to an opening 2212 in the cover plate 440.The micro valve has a fixed body 2214 with a membrane 2216 disposed onthe surface there above to define a pumping chamber 2218. The pumpingchamber 2218 has a hole 2220 interfacing the pumping chamber 2218 withthe opening 2208 on the cover plate 440. Similarly, the hole 2212 isinterfaced to the pumping chamber 2218 through a hole 2222. The membrane2216 is operable to reciprocate away from the surface of the fixed body2214 exposing the top of the hole 2210 in the pumping chamber 2218 toallow fluid to flow through the pumping chamber 2218 and down throughthe opening 2222 through the cover plate 440 and through to themicrochannel 2204. In the closed position, the membrane 2216 is forceddown against the upper end of the hole 2220. A pneumatic cavity 2230 isdisposed above the membrane 2216 in a housing 2232 and interfaces with apneumatic source through a hose 2234. Thus, by drawing a vacuum in thepneumatic cavity 2230, the membrane 2216 will be pulled away from thehole 2220 to allow fluid to flow and, when pressurized air is forcedinto the pneumatic cavity 2230, and the membrane 2216 is forced down tothe surface of the fixed body 2214 to seal the opening 2224 in a closedposition.

It will be appreciated by those skilled in the art having the benefit ofthis disclosure of a microfluidic testing system with cellcapture/analysis regions for processing a parallel and serial manner. Itshould be understood that the drawings and detailed description hereinare to be regarded in an illustrative rather than a restrictive manner,and are not intended to be limiting to the particular forms and examplesdisclosed. On the contrary, included are any further modifications,changes, rearrangements, substitutions, alternatives, design choices,and embodiments apparent to those of ordinary skill in the art, withoutdeparting from the spirit and scope hereof, as defined by the followingclaims. Thus, it is intended that the following claims be interpreted toembrace all such further modifications, changes, rearrangements,substitutions, alternatives, design choices, and embodiments.

What is claimed is:
 1. A microfluidic chip system comprising: a firstreservoir for holding a biologic sample containing a predeterminedbiologic material; a first plurality of parallel pathways for testing atreatment agent of a plurality of treatment agents and determining atreatment efficacy for the predetermined biologic material; a firstmicro-pump for pumping a portion of the biologic sample into each of thefirst plurality of parallel pathways from the first reservoir; aplurality of second reservoirs for holding the portion of the biologicsample treated with one of the plurality of treatment agents; a secondplurality of parallel pathways each for determining a dosage level of aparticular one of the plurality of treatment agents with respect to thepredetermined biologic material; and a plurality of second micro-pumpseach associated with one of the second plurality of parallel pathwaysfor pumping a second portion of the biologic sample into a selected oneof the second plurality of parallel pathways indicating the treatmentagent providing a best treatment efficacy of the predetermined biologicmaterial.
 2. The microfluidic chip system of claim 1, wherein the firstplurality of parallel pathways further comprises: a plurality ofmicro-channels, each of the plurality of micro-channels interconnectingthe first reservoir to the plurality of second reservoirs, each of theplurality of micro-channels having a portion thereof interiorly coatedwith one of the plurality of treatment agents for applying the treatmentagent to the predetermined biologic material passing through the portionof the micro-channel.
 3. The microfluidic chip system of claim 2,wherein each of the plurality of micro-channels include a serpentineportion for the portion of the micro-channel.
 4. The microfluidic chipsystem of claim 2, wherein the first micro-pump further pumps theportion of the biologic sample through the plurality of micro-channelsinto the plurality of second reservoirs.
 5. The microfluidic chip systemof claim 1, further comprising a first reading window for enabling adetection of the predetermined biologic material within the biologicsample.
 6. The microfluidic chip system of claim 5, further including asecond plurality of reading windows each associated with one of theplurality of second reservoirs for enabling a view of effects caused byapplication of the treatment agent to the biologic sample.
 7. Themicrofluidic chip system of claim 5, further comprising a cell counterassociated with the first reading window for applying an affinity labelto cells of the predetermined biologic material within the biologicsample.
 8. The microfluidic chip system of claim 1, wherein each of thesecond plurality of parallel pathways further comprises a plurality oftesting modules each for applying a different dosage level of one of theplurality of treatment agents to the predetermined biologic materialwithin the biologic sample.
 9. The microfluidic chip system of claim 8,wherein each of the plurality of testing modules further comprises: athird reservoir for holding the second portion of the biologic sampletreated with one of the plurality of treatment agents; and amicro-channel interconnecting the first reservoir to the thirdreservoir, the micro-channel including a portion interiorly coated withone of the plurality of treatment agents for applying the treatmentagent to the predetermined biologic material passing through themicro-channel at the different dosage level.
 10. The microfluidic chipsystem of claim 9, wherein the plurality of testing modules is connectedin series to test an efficacy of a plurality of dosage levels of thetreatment agent providing the best treatment efficacy one at a time. 11.The microfluidic chip system of claim 9, wherein the plurality oftesting modules is connected in parallel to test an efficacy of aplurality of dosage levels of the treatment agent providing the besttreatment efficacy at a same time.
 12. The microfluidic chip system ofclaim 1, wherein one of the plurality of second micro-pumps pump thesecond portion of the biologic sample into the selected one of thesecond plurality of parallel pathways in response to a control input.13. The microfluidic chip system of claim 1, further comprising an inputfor receiving the biologic sample.
 14. A method comprising: receivingand holding a biologic sample containing a predetermined biologicmaterial within a first reservoir of a microfluidic chip device; pumpinga portion of the biologic sample into each of a first plurality ofparallel pathways from the first reservoir using a micro-pump; applyinga treatment agent of a plurality of treatment agents within each of thefirst plurality of parallel pathways to the portion of the biologicsample within the parallel pathway; holding the portion of the biologicsample treated with one of the plurality of treatment agents in aplurality of second reservoirs; pumping a second portion of the biologicsample into a selected second parallel pathway, associated with aselected treatment agent of the plurality of treatment agents, of asecond plurality of parallel pathways from the first reservoir using asecond micro-pump; and applying the selected treatment agent at aplurality of different dosage levels within the selected second parallelpathway to the second portion of the biologic sample within the selectedsecond parallel pathway.
 15. The method of claim 14, wherein the step ofapplying the treatment agent further comprises pumping the biologicsample through a plurality of micro-channels interconnecting the firstreservoir with the plurality of second reservoirs to apply the pluralityof treatment agents, wherein one of the plurality of treatment agentsare applied in each of the plurality of micro-channel s.
 16. The methodof claim 14, wherein the step of applying the selected treatment agentfurther comprises applying the selected treatment agent in series at theplurality of different dosage levels to test an efficacy of theplurality of different dosage levels one at a time.
 17. The method ofclaim 14, wherein the step of applying the selected treatment agentfurther comprises applying the selected treatment agent at the pluralityof different dosage levels in parallel to test an efficacy of theplurality of different dosage levels at a same time.
 18. The method ofclaim 14, further comprising the step of applying an affinity label tocells of the predetermined biologic material within the biologic sampleusing a cell counter.
 19. The method of claim 14, further comprisingenabling detection of the predetermined biologic material using a firstreading window of the microfluidic chip device.
 20. The method of claim14, wherein pumping the second portion of the biologic sample into theselected second parallel pathway is responsive to a control input.